The immunological importance of the non-human Gal-α-(1→3)-Gal epitope was initially highlighted by its direct involvement in hyperacute rejection (HAR) of porcine organ xenografts. Xenotransplantation has been widely considered as a direct means of overcoming the critical shortage of human donor organs with porcine organs, in particular, being considered the most suitable. However, porcine tissue expresses a high proportion of the Gal-α-(1→3)-Gal epitope and elicits a vigorous anti-Gal-α-(1→3)-Gal antibody response following transplantation of porcine xenografts into humans. Undesirable immune responses to this epitope are also thought to be involved in the sub-optimal clinical outcomes after implantation of a cellular tissue matrices. Naturally-occurring human antibodies directed against the Gal-α-(1→3)-Gal epitope are widespread in man, comprising up to 3% of immunoglobulin (Ig) in human sera (mainly IgG2 subclass). Their induction by environmental stimuli, commensal bacteria and/or parasitic interaction has been proposed and they putatively exert a natural barrier function. As well as its involvement in HAR, the presence of this antigenic glycan moiety has also been attributed to an IgE-mediated allergic/hypersensitivity response observed in patients taking the recombinant monoclonal antibody preparation, Erbitux (Cetuximab). The levels of Gal-α-(1→3)-Gal epitope required to induce anaphylaxis remain undetermined and may be both product-specific and patient-dependent.
The production of recombinant antibodies and related biopharmaceuticals is now seen as a well-established and a routinely performed industrial process, as evidenced by the increase in biosimilar production plants over recent years. Control over glycosylation is a critical factor during recombinant therapeutic production because of its profound effect on protein function, allergenic and immunogenic properties, plasma clearance rates and efficacy. It is now accepted that specific glycan structures adversely affect the safety of these recombinant products. The mammalian cell lines utilised for production, most commonly murine derived cell lines SP2/0 and NSO or Chinese hamster ovary cells (CHO) permit ‘human-like’ glycosylation to occur. However, these cell line systems possess the molecular machinery to incorporate non-human glycan structures, including the Gal-α-(1→3)-Gal and N-glycolylneuraminic acid (Neu5Gc) moieties, into the target protein making them immunogenic in humans. The ability of the CHO system to synthesise the Gal-α-(1→3)-Gal glycan has only recently been reported, contrary to previously accepted reports detailing the lack of the biosynthetic machinery to synthesize glycoproteins with Gal-α-(1→3)-Gal moieties. Bosques et at demonstrated both the ability of CHO cells to synthesise the Gal-α-(1→3)-Gal moiety and its presence on the commercial therapeutic protein, Abatacept (Orencia), a CHO-generated antibody fusion protein. However, the level of Gal-α-(1→3)-Gal detected on proteins produced in the CHO system were lower than those typically observed on products derived from murine cell lines (Bosques et al., 2010). With the identification of Gal-α-(1→3)-Gal on recombinant proteins currently on the market, there is a need for convenient and rapid analytical approaches to monitor and quantify the levels of Gal-α-(1→3)-Gal on existing and future recombinant therapeutics for human use.
Furthermore, the possibility of exploiting the Gal-α-(1→3)-Gal epitope/natural human antibody system to improve the efficacy of autologous vaccines is gaining increased attention recently and this will also demand convenient assay tools.
The combination of high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) and endoglycosidase digestion provides sufficient resolution and sensitivity for the identification and measurement of N-glycans. In addition, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has also been used for profiling of the fluorescently labelled N-glycans because of its throughput and high-resolution separation capability. There have also been reports of structural analysis of N-glycans of a number of glycoproteins and recombinant mAbs by CE-LIF after fluorescent labelling with 8-aminopyren-1,3,6-trisulfonate (APTS). The problem with these methods is that they are cumbersome, time-consuming to perform and require specialist equipment and instrumental expertise.
A number of pathogenic organisms also express the Gal-α-(1→3)-Gal epitope, such as Trypanosoma cruzia, the causitive agent of Chagas' disease, American Leishmania, Colletotrichum, which is a fungal plant pathogen and Neisseria meningitides. Thus, an anti-Gal-α-(1→3)-Gal antibody or fragment could be used either in diagnosis of these diseases or in their therapy. This epitope has also been implicated in anaphylactic reactions to oligosaccharides found in red meat and thus the antibodies of the invention could find use in the prevention or prediction of such responses.
Specific binding assays provide robust analytical platforms once high affinity and specific binding agents are available for the analytes. Lectins are the most widely used affinity reagents for carbohydrates, with less reliance on antibodies, due to limited availability of high quality antibodies. Two lectins are commonly used for the detection of the Gal-α-(1→3)-Gal motif. Griffonia simplicifolia I isolectin B4 (GS-I-B4) detects terminal Gal-α-1-R (alpha-galactosyl residues, termed alpha-Gal or αGal) epitopes, but cannot distinguish between structures in which the terminal galactose is linked to different carbon atoms in the penultimate galactose on the carbohydrate chain (e.g. Gal-α-1>2, Gal-α-1→3 or Gal-α-1→4). Binding of GS 1-B4 may also depend on whether the Gal-α-(1→3)-Gal is on a cell surface or on an isolated glycoprotein, as has been reported for a number of glycan recognition molecules. Marasmius oreades agglutinin (MOA) is also known to bind with Gal-α-1-R terminated structures. Although a number of anti-Gal-α-(1→3)-Gal antibodies have been described, only a small number are commercially available, including a polyclonal antibody raised in baboon and the M86 mouse IgM monoclonal antibody which have found limited application to date.
There are still significant challenges in the generation of high quality antibodies targeting carbohydrate motifs because of their low immunogenicity. Yet, human serum and the serum of many animals contain a wide range of natural anti-carbohydrate antibodies. Engineered single chain antibody fragment (scFv) libraries generated from immunoglobulin cDNA, whether from naive or immune-challenged host systems, may provide access to these antibodies. Chickens, like humans, do not produce the Gal-α-(1→3)-Gal epitope and hence develop a strong immune response on exposure to this motif. The generation of chicken antibody libraries has been shown to be simpler than libraries from mammalian species, due to the peculiar mechanism of immunoglobulin gene diversification in birds. Chickens possess single functional immunoglobulin heavy chain variable region (VH) and light chain variable region (VL) genes, with diversity created by the high frequency gene conversion mechanism operating continuously during B cell proliferation in the bursa. Here we describe the generation of a Gal-α-(1→3)-Gal targeted phage displayed-scFv library and isolation of chicken scFv antibody fragments directed against this epitope. These scFvs were shown to be highly specific for the detection of the Gal-α-(1→3)-Gal motif when tested in direct ELISA format against a panel of related neoglycoconjugates (NGCs). The antibody fragments in of the present invention were demonstrated to have high affinity and specificity for the Gal-α-(1→3)-Gal motif and have thus proved to be more effective and therefore much more commercially useful than previously known anti-Gal-α-(1→3)-Gal antibodies. The scFvs of the invention were also used in competitive ELISA format, where they allowed the concentration of Gal-α-(1→3)-Gal to be determined in free solution and when present on the surface of a glycoprotein. To our knowledge, this is the first report of a panel of engineered scFvs against the non-human carbohydrate Gal-α-(1→3)-Gal motif, and most importantly the first report of a convenient competitive ELISA for detection of this motif on glycoproteins.